Phospholipase C-β and ovarian sex steroids in pig granulosa cells

We compared the membrane effects of estradiol, progesterone, and androstenedione in a single experimental model, the ovarian granulosa cells collected from immature Large White sows. We measured changes in cytosolic free calcium concentration ([Ca2+]i) in confluent Fura‐2 loaded cells. We used pharmacological tools and polyclonal phospholipase C‐β (PLC‐β) antibodies. Each steroid (0.1 pM to 1 nM) transiently increased intracellular calcium concentration ([Ca2+]i) within 5 sec. They mobilized Ca2+ from the endoplasmic reticulum as shown by using two phospholipase C inhibitors, neomycin and U‐73122. Ca2+ mobilization involved PLC‐β1 for progesterone, PLC‐β2 for estradiol and PLC‐β4 for androstenedione. A pertussis toxin‐insensitive G protein was involved in the effects of progesterone on Ca2+ mobilization whereas estradiol and androstenedione effects were mediated via a pertussis toxin‐sensitive G‐protein. Ca2+ influx from the extracellular milieu was involved in the increase in [Ca2+]i induced by progesterone and estradiol, but not by androstenedione. Influx of Ca2+ was independent of Ca2+ mobilization from calcium stores, and it was suggested that L‐type Ca2+ channels for estradiol and T‐type Ca2+ channels for progesterone were involved. The three steroids had no effect on cAMP. Rapid effects of progesterone, estradiol, and androstenedione involved a direct action on cell membrane elements such as PLC‐β, G‐proteins, and calcium channels, and these mechanisms were hormone‐specific. J. Cell. Biochem. 74:50–60, 1999. © 1999 Wiley‐Liss, Inc.